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A 2-aza-Cope reactivity-based platform for ratiometric fluorescence imaging of formaldehyde in living cells.

ABSTRACT: Formaldehyde (FA) is a major reactive carbonyl species (RCS) that is naturally produced in living systems through a diverse array of cellular pathways that span from epigenetic regulation to the metabolic processing of endogenous metabolites. At the same time, however, aberrant elevations in FA levels contribute to pathologies ranging from cancer and diabetes to heart, liver, and neurodegenerative diseases. Disentangling the complex interplay between FA physiology and pathology motivates the development of chemical tools that can enable the selective detection of this RCS in biological environments with spatial and temporal fidelity. We report the design, synthesis, and biological evaluation of ratiometric formaldehyde probe (RFAP) indicators for the excitation-ratiometric fluorescence imaging of formaldehyde production in living systems. RFAP-1 and RFAP-2 utilize FA-dependent aza-Cope reactivity to convert an alkylamine-functionalized coumarin platform into its aldehyde congener with a ca. 50 nm shift in the excitation wavelength. The probes exhibit visible excitation and emission profiles, and high selectivity for FA over a variety of RCS and related reactive biological analytes, including acetaldehyde, with up to a 6-fold change in the fluorescence ratio. The RFAP indicators can be used to monitor changes in FA levels in biological samples by live-cell imaging and/or flow cytometry. Moreover, RFAP-2 is capable of visualizing differences in the resting FA levels between wild-type cells and models with a gene knockout of ADH5, a major FA-metabolizing enzyme, establishing the utility of this ratiometric detection platform for identifying and probing sources of FA fluxes in biology.

SUBMITTER: Brewer TF

PROVIDER: S-EPMC5434806 | biostudies-other | 2017 May

REPOSITORIES: biostudies-other

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A 2-aza-Cope reactivity-based platform for ratiometric fluorescence imaging of formaldehyde in living cells.

Brewer Thomas F TF Burgos-Barragan Guillermo G Wit Niek N Patel Ketan J KJ Chang Christopher J CJ

Chemical science 20170407 5

Formaldehyde (FA) is a major reactive carbonyl species (RCS) that is naturally produced in living systems through a diverse array of cellular pathways that span from epigenetic regulation to the metabolic processing of endogenous metabolites. At the same time, however, aberrant elevations in FA levels contribute to pathologies ranging from cancer and diabetes to heart, liver, and neurodegenerative diseases. Disentangling the complex interplay between FA physiology and pathology motivates the dev ...[more]

PMID: 28580121

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Project description:Potassium is the most abundant intracellular metal in the body, playing vital roles in regulating intracellular fluid volume, nutrient transport, and cell-to-cell communication through nerve and muscle contraction. On the other hand, aberrant alterations in K+ homeostasis contribute to a diverse array of diseases spanning cardiovascular and neurological disorders to diabetes to kidney disease to cancer. There is an unmet need for studies of K+ physiology and pathology owing to the large differences in intracellular versus extracellular K+ concentrations ([K+]intra = 150 mM, [K+]extra = 3-5 mM). With a relative dearth of methods to reliably measure dynamic changes in intracellular K+ in biological specimens that meet the dual challenges of low affinity and high selectivity for K+, particularly over Na+, currently available fluorescent K+ sensors are largely optimized with high-affinity receptors that are more amenable for extracellular K+ detection. We report the design, synthesis, and biological evaluation of Ratiometric Potassium Sensor 1 (RPS-1), a dual-fluorophore sensor that enables ratiometric fluorescence imaging of intracellular potassium in living systems. RPS-1 links a potassium-responsive fluorescent sensor fragment (PS525) with a low-affinity, high-selectivity crown ether receptor for K+ to a potassium-insensitive reference fluorophore (Coumarin 343) as an internal calibration standard through ester bonds. Upon intracellular delivery, esterase-directed cleavage splits these two dyes into separate fragments to enable ratiometric detection of K+. RPS-1 responds to K+ in aqueous buffer with high selectivity over competing metal ions and is sensitive to potassium ions at steady-state intracellular levels and can respond to decreases or increases from that basal set point. Moreover, RPS-1 was applied for comparative screening of K+ pools across a panel of different cancer cell lines, revealing elevations in basal intracellular K+ in metastatic breast cancer cell lines vs. normal breast cells. This work provides a unique chemical tool for the study of intracellular potassium dynamics and a starting point for the design of other ratiometric fluorescent sensors based on two-fluorophore approaches that do not rely on FRET or related energy transfer designs.

Dataset Information

A 2-aza-Cope reactivity-based platform for ratiometric fluorescence imaging of formaldehyde in living cells.

Publications

A 2-aza-Cope reactivity-based platform for ratiometric fluorescence imaging of formaldehyde in living cells.

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